Automated system for the lysis of microorganisms present in a sample, for extraction and for purification of the nucleic acids of said microorganisms for purposes of analysis

ABSTRACT

A method for collecting microorganisms when contained in a fluid includes (i) introducing the fluid into a cavity of a collecting device via at least one admission duct, (ii) capturing the microorganisms when contained in the fluid with a set of beads retained in the cavity as the fluid passes through the set of beads, (iii) evacuating the fluid from the cavity via at least one evacuating duct, (iv) introducing a reaction liquid into the cavity via at least one admission channel, (v) collecting the microorganisms from the set of beads with the reaction liquid as the reaction liquid passes through the set of beads, and (vi) evacuating the reaction liquid from the cavity via at least one evacuating channel.

This is a Divisional of application Ser. No. 14/409,979 filed Dec. 19,2014, which is a National Stage Entry of PCT/FR2013/051681 filed Jul.12, 2013, which claims priority to FR 1256758 filed Jul. 13, 2012. Thedisclosure of the prior applications is hereby incorporated by referenceherein in its entirety.

The field of technology of the present invention is that of biologicalanalysis. More particularly, the present invention relates firstly to adevice for lysis of the microorganisms present in an environmental orclinical sample, for extraction and for purification of the nucleicacids of said microorganisms. The invention further relates to anautomated system for lysis of microorganisms for extraction andpurification of the nucleic acids of said microorganisms, for thepurposes of analysis.

For several years there has been a considerable increase in theincidence of nosocomial infections in hospitals. These infections areexplained by the contamination of the hospitalized persons, whotherefore are by definition immunodepressed, by pathogenicmicroorganisms that are present in the hospital environment and are notdestroyed despite the great care always paid to disinfection ofequipment and surfaces and to air conditioning. In view of these moreand more frequent cases of environmental microbiological contamination,development of devices and methods for improving and facilitatingenvironmental controls has become a major challenge for healthprofessionals.

Apart from the problem of nosocomial infections, monitoring ofenvironmental conditions has over recent years been a recurrent concernin the industrial environment, in particular in the food industries andthe pharmaceutical or cosmetics industries. In the food industries, weare familiar with the disastrous consequences that contamination ofproducts, or even of raw materials, with a pathogenic microorganism mayhave for consumers' health. In fact, toxic food infections, due tobacteria such as those of the genus Listeria or Salmonella, are now ofcommon occurrence. Monitoring of air quality is also a key process inthe quality approach of the pharmaceutical or cosmetics industries.

Moreover, this monitoring must meet an ever increasing level ofrequirements, owing to increasingly stringent regulations.

Among the tools available to health professionals or manufacturers forcarrying out environmental monitoring, the air biosamplers are solutionsof choice for detecting microorganisms in the air. These devices areplaced at suitable points in places where we wish to measureaero-bio-contamination. They generally consist of an air sampler coupledto a culture medium. The air collected by the air sampler comes intocontact with the culture medium; any airborne microorganisms collectedwill be deposited on said culture medium. The culture medium is thenrecovered and put in a stove to promote growth of the microorganisms. Itis thus possible to detect and identify said microorganisms bytraditional microbiology techniques.

These devices nevertheless have a major drawback that is associated withthe technology used. This drawback is the time taken to obtain theanalysis result. In fact, the use of the traditional techniques ofmicrobiology, especially of bacteriology, involves waiting theincubation times necessary for cellular growth, or even for steps ofreseeding on specific culture media to allow identification. As aresult, the time taken to obtain a result is relatively long, or eventoo long, when we wish to detect and identify a pathogenic organismresponsible for a nosocomial infection or for food poisoning.

Another drawback of this type of device is that although the use ofculture media makes it possible to discriminate between genera andspecies of bacteria, it does not generally make it possible todiscriminate between the strains of one and the same bacterial species.Now, it is known that the pathogenicity of a microorganism may varysignificantly depending on the strain in question.

Moreover, this type of device has the drawback that it does not allowdetection of airborne microorganisms that are viable but are notcultivatable.

These devices also have the drawback of drying of the nutrient surfacesas collection continues.

Moreover, there are devices that are intended for recovery of airborneparticles, especially microorganisms. Thus, document GB-2 254 024describes a device for collecting the particles present in the air basedon the principle of the cyclone effect. Although such a device provessuitable for collecting the particles present in the air, includingmicroorganisms, it has never been investigated for treating the samplethus obtained, in particular for carrying out extraction of the geneticmaterial, intended to be used for analysis.

More generally, the most relevant techniques in terms of identificationof microorganisms and/or of speed of delivering the results, whetherwith respect to clinical samples or environmental samples, are withoutany doubt the techniques of molecular diagnostics. These techniques,based on analysis of the genetic material of the microorganisms, and inparticular of certain specific sequences of interest, make it possibleto obtain very precise identification of the microorganisms in a recordtime, since they make it possible to omit the culture steps.

Nevertheless, the use of such techniques has a certain number oflimitations, the most important of which is the potentially limitedquantity of microorganisms present in the air and therefore recoverablefor performing the analysis. In fact, it is known that environmentalsamples, but also certain clinical samples, are relatively poor inmicroorganisms. As a result, the amount of genetic material obtainedfrom this raw material is small. The performance of the technique usedfor extracting the nucleic acids, in terms of yield, then becomes acrucial parameter.

Moreover, most of the existing techniques for lysis of microorganismsare not general with respect to all microorganisms and/or require theintervention of qualified personnel for performing the manual steps.

Document WO-A-2005/038025 describes a method for extracting nucleicacids from microorganisms notably sampled from the air. This methodconsists of employing three different methods of lysis, namely chemicallysis, heat shock lysis and mechanical lysis. If such a method makes itpossible without any doubt to optimize the efficiency of extraction ofthe nucleic acids and therefore increase the amount of genetic materialavailable for analysis, it is still the case that this efficiencyremains dependent on the quantity of microorganisms recovered. Now,nothing is described in this document for optimizing the recovery ofsaid microorganisms.

Document U.S. Pat. No. 5,707,861 describes a device for disruptingliving cells such as microorganisms. This device allows cells to belysed using both glass beads and the effect of vibration due to thespace between the tubes containing the microorganisms and the holes ofthe support carrying said tubes. Thus, such a device makes it possibleto optimize cellular lysis and therefore optimize extraction of thegenetic material. Such a device and the method that it employs have thesame limitations as those discussed above, namely that they remaindependent on the quantity of microorganisms recovered. Moreover, theyhave the additional drawback of requiring a subsequent step ofconcentration of the nucleic acids in order to isolate them from thecellular debris. Finally they require recovering the nucleic acidsmanually at the end of the concentration step.

These problems also arise with the device described in document U.S.Pat. No. 5,567,050.

Systems with greater integration have also been described. Thus,document WO-A-2004/018704 describes a device, and an associated methodusing the technique of PCR (Polymerase Chain Reaction) amplification,for collecting microorganisms in the air and identifying them. Thissystem is specially designed for combating assassination attempts bybiological contamination in mail sorting centers. This system consistsof an air sampling device placed along the circuit for transporting themail, a device for filtration/separation of particles by the cycloneeffect, a device for concentration/recovery of particles from a liquidsample, a device for transferring a fraction of the sample into aGeneXpert™ PCR analysis cartridge from the company Cepheid inCalifornia. The cartridge is then transferred manually to an independentautomatic biological analyzer for identification of the microorganism ormicroorganisms collected from the air.

Although this system solves a good number of technical problemsassociated with the devices and methods described above, it neverthelesshas major drawbacks. The first of these drawbacks is that the system forsample treatment (collection, separation, concentration/recovery) priorto transfer to the analysis cartridge is relatively complex andexpensive. A second drawback is that the microorganisms collected arerecovered in a liquid sample, only a fraction of which is analyzed. Thismeans that there is a considerable risk of not recovering all of themicroorganisms and therefore all of the nucleic acids, which greatlylimits the suitability of the analysis. Moreover, despite itscomplexity, this system requires manual transfer of the cartridge intothe GeneXpert™ automatic analyzer.

The applicant has already filed a patent application WO-A-2009/001010that relates firstly to a cartridge, suitable for placing inside an airsampling means and for receiving a means for recovering the nucleicacids, said cartridge, of roughly cylindrical shape, comprising aretention zone of the microorganisms, said retention zone comprisingmeans for lysis of microorganisms. This invention further relates to adevice for collecting airborne microorganisms and a device for lysis ofmicroorganisms.

The applicant has also already filed a patent applicationWO-A-2010/067019 which, among other things, relates to a device forcollecting airborne microorganisms, said device comprising:

-   -   an air sampling module, comprising:    -   i. an upper element comprising an air admission duct allowing        entry of a stream of air into said module, said duct having, at        its base, means for disturbing the air stream,    -   ii. a lower element comprising means for evacuating the air,        allowing exit of the air stream created        -   it being possible for said upper and lower elements to be            integrated with one another so that the air stream can be            created within said air sampling module;    -   a cartridge, of roughly cylindrical shape, comprising a        retention zone of the microorganisms, said retention zone        comprising means for lysis of microorganisms, said cartridge        being positioned inside said air sampling module.

With these two novel solutions, it is undeniable that the process isimproved with universal lysis, effective both for environmental andclinical samples, for a great variety of microorganisms, whether theyare bacteria, viruses or fungi, optionally in the vegetative state or inthe form of spores.

Moreover, there is quite a high concentration of microorganisms, thesystem is compatible with various downstream analyses (such as PCR,streaking on dishes, etc.), and the mechanical lysis is integrated.

However, it is not in human nature to be content with a system thatfunctions well, when an even more optimal solution may be found. Thus,in these two instances, collection is still limited by the volume offluid, gaseous or liquid, which comes into contact with the recoverymedium, generally a culture medium. The stream of fluid will alwaysimpinge on this medium at the same point, which will lead:

-   -   either to saturation of said medium in a precise spot without        the rest being used,    -   or to degradation of this medium as it is always impacted by a        gas or a liquid at the same point(s), which leads to desiccation        in the case of a gas and liquefaction of the surface of the        medium in the case of a liquid. In both cases and notably in the        case of desiccation of agar, there will be a very marked        decrease in capture efficiency as a function of time.

Moreover, the manner of capture by impacting is very inefficient forsmall particles with a diameter of less than 1 μm. Another problem isthat there is a risk of entrainment of a proportion of the recoverymedium (beads with glycerol) by the air stream. Thus, this projection offluid and its rebound on said medium may generate splashes, which are afurther risk of contamination.

The aim of the present invention is to propose an apparatus that tacklesthe drawbacks described above.

For this, the invention proposes a device for collecting microorganismscontained in a fluid, said device comprising:

-   -   a reaction module containing a set of beads,    -   at least one fluid admission duct allowing a stream of fluid to        enter the module,    -   at least one duct for evacuating the fluid, allowing exit of the        stream of said fluid that has passed through the interior of        said module,    -   means for retaining the beads inside the module,    -   at least one channel for admitting at least one reaction liquid,    -   at least one channel for evacuating at least one reaction        liquid,    -   the channels for admitting and evacuating the reaction liquid        and the ducts for admission and evacuation of the fluid being        positioned as follows at module level:    -   the ducts for admission and evacuation of the fluid facing one        another while enclosing the module, in order to maximize        contacts between the fluid and the beads,    -   the channels for the reaction liquid or liquids being positioned        respectively at two opposite ends of the module, and    -   the channels for the reaction liquid or liquids are positioned        in one plane, and the ducts for admission and evacuation of the        fluid are positioned along an axis, said axis being roughly        perpendicular to the plane.

According to a first embodiment, the device according to the inventioncomprises an airtight confinement enclosure, isolating the fluid and thereaction liquids from the exterior.

According to a first embodiment of the device, if the fluid is a gas,the set of beads consists of beads coated with glycerol.

According to a second embodiment of the device, if the fluid is aliquid, the module contains a filter extending approximately in theplane formed by the channels for the reaction liquid or liquids, theadmission channel(s) being positioned on one side of the filter with theset of beads and the evacuating channel(s) being positioned on the otherside of said filter in the absence of beads. This arrangement thusallows the fluid to come into contact with the beads and then circulatethrough the filter, in order to separate for example any cellularresidues that are of no interest or inhibitors, such as proteins,membranes.

In a particular embodiment, the shape of the module, if it is cut:

-   -   along the plane formed by the channels for admitting and        evacuating the reaction liquid, and    -   perpendicularly to the axis formed by the ducts for admission        and evacuation of the fluid,

comprises at least one rectilinear portion and/or at least one portionin an arc, which are comprised between two ends. The channel(s) foradmission of the reaction liquid are located at one end of the moduleand the channel(s) for evacuating the reaction liquid are located at theother end of the module, so that the reaction liquid passes completelythrough the bed of beads between the moment when it is admitted into theadmission channel(s) of the reaction liquid and the moment when it isevacuated via the evacuating channel(s) of the reaction liquid.

According to this last-mentioned embodiment or variant, the module has aquadrilateral section if it is cut along a plane perpendicular to theplane formed by the channels for admitting and evacuating the reactionliquid, and passing along the axis formed by the ducts for admission andevacuation of the fluid.

In a particular embodiment, the module is of “C” shape if it is cut:

-   -   along the plane formed by the channels for admitting and        evacuating the reaction liquid, and    -   perpendicularly to the axis formed by the ducts for admission        and evacuation of the fluid

A “C” shape comprises at least one portion in an arc corresponding to aportion of the circumference of a circle or of a parabola joining twopoints. The ends of the “C” being the two end zones of the module, sothat the channel(s) for admission of the reaction liquid are located atone end of the module and the channel(s) for evacuating the reactionliquid are located at the other end of the module, the reaction liquidpasses completely through the bed of beads between the moment when it isadmitted into the admission channel(s) of the reaction liquid and themoment when it is evacuated via the evacuating channel(s) of thereaction liquid.

According to this last-mentioned embodiment or variant, the module is ofquadrilateral shape if it is cut along a radius passing through thecenter of the “C” and cutting through said “C”.

Still according to this last-mentioned embodiment or variant, theadmission channel(s) of the reaction liquid is(are) located at one endof the “C” of the shape of the module, and the evacuating channel(s) ofthe reaction liquid is(are) located at the other end of the “C”.

Regardless of the embodiment or variant, the beads have a diameterbetween 200 and 600 μm and the means for retaining said beads inside themodule consist of grids, the pores of which have a diameter smaller thanthe beads and in the range from 100 to 500 μm.

Regardless of the embodiment or variant, it comprises another reactionmodule for treatment of the microorganisms collected, said modulecontaining:

-   -   means for separating the cellular residues that are of no        interest or inhibitors, such as proteins, membranes, and/or    -   means for amplification of the nucleic acids collected and        separated from the inhibitors, and/or    -   means for detecting the amplicons thus generated.

The present invention also relates to a method for collectingmicroorganisms contained in a fluid, which consists of:

-   -   introducing the fluid suspected of containing microorganisms via        at least one fluid admission duct,    -   passing through a bed of beads, where the microorganisms are        immobilized,    -   evacuating the fluid, from which said microorganisms have been        removed, via at least one duct for evacuating said fluid,    -   introducing at least one reaction liquid via at least one        reaction liquid admission channel,    -   passing through the bed of beads where the microorganisms are        immobilized in order to put said microorganisms in suspension,        and    -   evacuating this liquid transporting the microorganisms via at        least one channel for evacuating the reaction liquid(s),    -   the ducts for admission and evacuation of the fluid facing one        another while enclosing the bed of beads, the channels for        admitting and removing the reaction liquid(s) being positioned        so as to reach all of the beads.

According to one embodiment, when the fluid is a gas, the beads arecoated with glycerol.

According to one embodiment, when the fluid is a liquid, the beads arecoated with a polymer coating.

According to one embodiment, when the reaction liquid passes through thebed of beads, said beads are agitated by an external agitating means andcome into contact, which makes it possible to destroy or to disrupt themembranes of the microorganisms, making the nucleic acids accessible andready to be evacuated.

According to one embodiment, a defined amount of reaction liquid isadmitted into the admission channel(s) of the reaction liquid and is notevacuated via the evacuating channel(s) of the reaction liquid duringlysis, so that the microorganisms are lysed for a defined time in thepresence of a defined amount of reaction liquid. This time is preferablybetween 5 and 30 minutes and this amount of liquid is preferably between100 μl and 1 ml, and more preferably is 500 μl.

According to a variant of this embodiment, the beads are agitated usingultrasound.

Regardless of the embodiment or variant, the liquid loaded with some orall of the microorganisms is evacuated to another reaction module of thedevice allowing treatment of the microorganisms collected, saidtreatment consisting of:

-   -   separating the cellular residues that are of no interest or        inhibitors, such as proteins, membranes, and/or    -   amplifying the nucleic acids collected, and/or    -   detecting the amplicons thus generated.

Regardless of the embodiment or variant, a first possibility for thismethod is characterized in that:

-   -   the fluid tested consists of a gas (for example air),    -   the gas passes through the bed of beads, said beads being coated        with glycerol, where the microorganisms are immobilized, and    -   at least one reaction liquid is introduced in order to liquefy        the glycerol and put said microorganisms in suspension.

Regardless of the embodiment or variant, a second possibility for thismethod is characterized in that:

-   -   the fluid tested consists of a liquid,    -   the liquid tested passes through the bed of beads and then a        filter before being evacuated, after removal of the        microorganisms that it contained; the microorganisms are        immobilized, and    -   at least one reaction liquid is introduced in order to put said        microorganisms in suspension.

According to either one of these two possibilities, the liquid isfiltered between evacuation via at least one channel for evacuating thereaction liquid and the module for removing the inhibitors and/or foramplification and/or for detection.

Fluid sample means any gaseous or liquid sample that may containmicroorganisms. It may be a sample of human or animal origin. Thissample may be, for example, urine, whole blood, plasma or any other bodyfluid or exhaled air. The sample may be of food origin such as a drink.It may also be of environmental origin, such as water or confined air.Moreover, the liquid sample may also be a so-called transfer liquid, inwhich any microorganisms contained on a surface sampling device, of theswab type such as those marketed by the company COPAN, under the name“flocked SWABS”, have been resuspended by agitating said swab in saidtransfer liquid.

The microorganisms are from the group comprising bacteria, viruses,yeasts, molds, parasites.

The samples from which the microorganisms are isolated are ofenvironmental origin. Thus, it may be a sample of air or of liquid, suchas water; surface samples. The samples may also be of clinical origin,namely any sample of human or animal origin, suitable as the object ofan analysis for detecting it and the identification of a microorganism,optionally pathogenic.

The presence of the target nucleic acids may be detected byvisualization of hybridization reactions. Hybridization reaction meansany reaction between a capture nucleic acid and a target nucleic acidisolated or generated by a step of transcription, reverse transcriptionor amplification, for example of the NASBA type (for Nucleic AcidSequence Based Amplification) or PCR.

Nucleic acid means oligonucleotides, deoxyribonucleic acids andribonucleic acids, and derivatives thereof. The term oligonucleotidedenotes a sequence of at least two nucleotides (deoxyribonucleotides orribonucleotides, or both), preferably at least five, preferably at leasteight, and even more preferably at least fourteen, whether they arenatural or modified, able to hybridize, in suitable hybridizationconditions, to another oligonucleotide, at least partiallycomplementary.

Modified nucleotide means for example a nucleotide comprising a modifiedbase and/or comprising a modification at the level of theinternucleotide bond and/or at the level of the skeleton. As an exampleof modified base, we may mention inosine, methyl-5-deoxycytidine,dimethylamino-5-deoxyuridine, diamino-2,6-purine andbromo-5-deoxyuridine.

To illustrate a modified internucleotide bond, we may mention thephosphorothioate, N-alkylphosphoramidate, alkylphosphonate andalkylphosphodiester bonds.

The alpha-oligonucleotides such as those described in patent applicationFR-A-2,607,507, the LNAs such as phosphorothioate-LNA and 2′-thio-LNAdescribed in Bioorganic & Medicinal Chemistry Letters, Volume 8, Issue16, Aug. 18, 1998, pages 2219-2222, and the PNAs that are the subject ofthe article by M. Egholm et al., J. Am. Chem. Soc. (1992), 114,1895-1897, are examples of oligonucleotides consisting of nucleotideswhose skeleton has been modified.

Visualization of the hybridization reactions may be carried out by anydetecting means, such as direct or indirect means.

In the case of direct detection, i.e. without the intermediary oflabeling, the hybridization reactions are observed by plasmon resonanceor by cyclic voltammetry on an electrode bearing a conductive polymer.

In the case of indirect detection, i.e. via labeling, the labeling maybe performed either directly on the target nucleic acids, or via aspecific binding partner of said previously labeled nucleic acids.

Specific binding partner of the target nucleic acids means any partnercapable of binding to the target nucleic acid, and as examples we maymention nucleic acids, oligonucleotides or polynucleotides and enzymesubstrates.

Labeling means fixation of a marker capable of directly or indirectlygenerating a detectable signal. A non-exhaustive list of these markersconsists of: enzymes that produce a signal that is detectable forexample by electrochemistry, colorimetry, fluorescence, luminescence,enzymes such as horseradish peroxidase (HRP), alkaline phosphatase(ALP), beta-galactosidase, glucose-6-phosphate dehydrogenase; enzymeinhibitors; enzyme co-factors; particles such as gold particles,magnetic latices, liposomes; chromophores such as luminescent compounds,dyes, radioactive molecules such as ³²P, ³⁵S or ¹²⁵I, fluorescentmolecules such as fluorescein, rhodamine, the Alexa®, umbelliferone,luminol or the phycocyanins. In the case of fluorescence, it may be thefluorescent product of an enzyme-substrate reaction, afluorophore-quencher combination, extinction of fluorescence or anyother system based on fluorescence properties.

Indirect systems may also be used, for example via anotherligand/antiligand pair. Ligand/antiligand pairs are well known by aperson skilled in the art, and we may mention for example the followingpairs: biotin/streptavidin, sugar/lectin, polynucleotide/complementarypolynucleotide. In this case, it is the ligand that bears the bindingagent. The antiligand may be detectable directly by the markersdescribed in the preceding paragraph or may itself be detectable by aligand/antiligand.

These indirect detection systems may lead, in certain conditions, toamplification of the signal. This technique for signal amplification iswell known by a person skilled in the art, and reference may be made tothe applicant's earlier patent applications FR-A-2 781 802 andWO-A-95/08000 or to the article J. Histochem. Cytochem. 45: 481-491,1997.

Preliminary labeling of the target nucleic acids may be carried out bydirect or indirect incorporation of a marker by a polymerase, by akinase, randomly or specifically, at the ends or by incorporation“inside” the molecule.

Labeling of the specific binding partners of the target analytes is wellknown by a person skilled in the art and is described for example byGreg T. Hermanson in Bioconjugate Techniques, 1996, Academic Press Inc,525B Street, San Diego, Calif. 92101 USA.

Depending on the type of labeling of the conjugate used, for exampleusing an enzyme, a person skilled in the art will add reagents forvisualization of the labeling. This step corresponds to development. Itis preceded by the use of a washing buffer that makes it possible toremove the fractions of analytes or of elements not used in thereaction, or bound weakly or nonspecifically, in order to limit thebackground noise.

The aims and advantages of the device according to the present inventionwill be better understood in light of the following example, not in anyway limiting, referring to the figures, in which:

FIG. 1 shows an exploded top view of the card or cartridge used forcollecting and lysing the microorganisms with all the elements thatallow it to function, according to a particular embodiment of theinvention.

FIG. 2 shows a perspective top view of the main card forming the core ofthe device according to the embodiment of the invention shown in FIG. 1.

FIG. 3 shows a perspective bottom view of the main card, still accordingto the embodiment of the invention shown in FIG. 1.

FIG. 4 shows a detail of FIG. 2 at the level of the bed of beads andgrid retaining them in the plane in contact with at least one fluidadmission duct and allowing capture of the microorganisms. The twoopposite ends of the C-shaped module are connected fluidically to thechannels for admitting and evacuating the reaction liquid.

FIG. 5A is a flowsheet of the filtering part of the main card where thebeads are present when the confining plates are closed.

FIG. 5B is a flowsheet similar to that of FIG. 5A, but in which theconfining plates are open.

FIG. 6 gives a detail A from FIG. 5B but in which the movement of thefluid within the capture beads and the movement of the confining platesare highlighted.

FIG. 7 is identical to FIG. 6 but focuses on the new movement of theconfining plates and the start of movement of the reaction liquid withinthe capture beads.

FIG. 8 is identical to FIG. 7 and clearly shows the movement of thefluid within said capture beads.

FIG. 9 is identical to FIG. 8 but emphasizes the extraction of thenucleic acids.

FIGS. 10A to 10D show the effectiveness of filling of the chamber wherethe microorganisms are retained by the retaining means over the courseof time.

FIG. 11 shows a graph demonstrating the efficiency of lysis within adevice according to the invention.

The present invention relates to a microorganism collecting device 1.FIG. 1 shows an exploded view of one embodiment presented by theapplicant in which the microorganism collecting device 1 is presentedwithout the presence of any fluid 2 or liquid 12. Said collecting device1 essentially comprises two main parts, a main card 4 positioned in alower position relative to a secondary card 5, which is in an upperposition. The main card 4 is L-shaped, and the secondary card 5 isintegrated in this “L” shape to give a general parallelepipedal shape.The main card 4 and secondary card 5 are intended to be brought intocontact with one another. In this way they will form a means 9 forretaining beads 6, essentially consisting of a cavity present in thehorizontal portion of the “L” shape, this cavity 9 being closed by thebottom face of the secondary card 5 in contact with card 4. The twocards, the main card 4 and the secondary card 5, once assembled form thereaction module 3. However, the retaining means 9 does not simplyconsist of the edges of this reaction module 3 but also comprises one ormore grids 27, not shown in FIG. 1, allowing passage of fluid 2 orliquids 12 but without allowing the beads 6, also not shown, to leavethe reaction module 3. On the grid 27, shown in FIG. 4, the diameter ofthe holes 28 in the grid 27 depends on the diameter of the beads 6 used.The holes 28 in the grid 27 must be of smaller diameter than thediameter of the glass beads. In a preferred embodiment, the holes 28 inthe grid 27 must be of significantly smaller diameter than the diameterof the glass beads, “significantly” meaning for example that the beads 6have a diameter from 400 to 600 μm and that the grid 27 has holes 28from 200 to 250 μm.

The assembly of the two cards 4 and 5 as well as the ducts for admissionand evacuation of the fluid 40 and 41 and channels for admitting andevacuating the reaction liquid 7 and 8, shown in FIG. 4, is confinedusing an airtight confinement enclosure 13, which consists, in theembodiment presented in FIG. 1, of a certain number of confining plates.

Firstly, in an upper position, there are two top plates 14 and 15. Thenin a lower position there are two bottom plates 16 and 17; plates 14 and16 provide confinement of the reaction module 3 notably at the level ofthe fluidic channels of the analysis zone 26, which will be describedlater. Then there are the top and bottom ferromagnetic confining plates15 and 17, which for their part are positioned above said reactionmodule 3.

After the capture step, plates 15 and 17 are repositioned to close theconsumable. Only the microorganisms 10 remain in the chamber 9. Channels7 and 8 are then used for circulating the elution buffer before theultrasonic lysis step.

It should be noted that the top plate 15 and the bottom plate 17 are ofa ferromagnetic nature as they enclose a certain number of magnets 21positioned at the four corners of the reaction module 3 as well as atits center. The magnets 21 provide, on the one hand, pressure of theferromagnetic plates 15 and 17 on the O-ring seals 18 and, on the otherhand, hermeticity after capture of the microorganisms. These plates 15and 17 provide access to the capture beads 6, and make it possible tocapture the microorganisms 10 present in the fluid 2 on the beads 6.Plates 15 and 17 slide over the surface of the device 1 on slides 29,located on the sides, and in the present case they are of dovetailshape. On completion of air sampling, said plates 15 and 17 arerepositioned to their original location by sliding along the slides 29.The liquid circuit is then closed. A “clamping” force is required on theplates in order to ensure perfect hermeticity on closure. This force isfor example ferromagnetic, which has the advantage of being a uniformpressure force on the entire surface of the plates in question.

Between the main card 4 and secondary card 5 and the top plate 15 andbottom plate 17, hermeticity is ensured by a set of hermetic O-ringseals 18. These O-ring seals 18 are three in number in the upperposition and three in the lower position and are concentric. Of course,this number is not in any way limiting since the use of a single O-ringseal may suffice. Moreover, other sealing means exist between the topplate 14 and bottom plate 16 and the rest of the device 1. The meansemployed are notably gluing of each of these plates with a glue or adouble-sided adhesive on the main card 4. Similarly, the secondary card5 is glued permanently on the body 4 of the device. This gluing isperformed ultrasonically, by laser or thermally, it is permanent andallows assembly of the collecting device 1.

It should be noted that the main card 4 comprises two zones where thereare channels. Firstly there is a storage zone referenced 25 near thereaction module 3. There is also an analysis zone 26, which wasmentioned above. The various channels present at the level of zones 25and 26 allow fluidic management of the whole card. The channel in thestorage zone 25 is the reservoir for storage of the reaction liquid 12(elution buffer). The channels in the analysis zone 26 are the fluidicchannels for sample preparation for analysis. They circulate on thefront and back of the card. Fluorescence reading also takes place in thechannels in zone 26. Since these zones 25 and 26 are not the essence ofthe invention, they will not be described further.

These types of channels and their functions are better explained in aprevious patent application WO-A-2011/033231 filed by the applicantunder French priority of Sep. 18, 2009. The reader is invited to referto this for fuller information.

After capture of the microorganisms 10, plates 15 and 17 are closedagain. The buffer from chamber 25 is sent into the capture chamber 9 forresuspending the microorganisms. Then ultrasonic lysis is carried out inthis same chamber 9 by a means external to the card. The lysate is thenshared via the distributor valve 22, which slides in the hole 31, and issent into the channels of zone 26 by means of the pump pistons 23, whichslide in holes 32, for preparation for detection and amplification ofthe nucleic acids 11.

However, it should be understood that various elements allow fluidicmanagement and transfer of the reaction liquid 12 from compartment tocompartment through the intervention of the distributor valve 22, aswell as the pump pistons 23. Three of these pump pistons 23 are on theleft of the main card 4, and the other three are on the right of thissame card 4.

Finally, to finish the description of FIG. 1, it is noted that there aredesiccating stations 24, still within the main card 4. These stations 24are located near the analysis zone 26, and contain drying agents foroptimizing storage of the reagents integrated with the card in the formof beads of lyophilized reagent, also called “pellets”, obtained bylyophilization, by drying or else by gelation. The drying agents are notin direct contact with but are close to the pellets. These pelletscontain the reagents necessary for amplification of the nucleic acids11.

The distributor valve 22 controls the distribution of the fluids in thecollecting device 1. Accordingly, there are several positions, whichprovide:

-   -   storage of the reaction liquid 12,    -   transfer of the reaction liquid 12 to the chamber or retaining        means 9 containing the beads 6 (after capture of the        microorganisms 10),    -   transfer of a total volume of 120 μl of the lysate, or 20 μl per        channel, to the six channels of zone 26.

Finally, the pump pistons 23 are similar to syringes and allowaspiration and transfer of the reaction liquid 12 throughout theprocess. Their particular feature is that they function in a closed aircycle (no air is aspirated from the exterior), although a simplerversion with aspiration of external air is conceivable. The benefit ofthis closed vessel is maximum avoidance of communication with theexterior and thus risk of cross contamination from the interior to theexterior and vice versa.

It can be seen in FIG. 1 that there is a spring plate 19 for locking theferromagnetic plates 15 and 17. This plate 19 is attached to thereaction module 3 by a screw 20. Thus, it should not be possible tomanipulate these detachable plates unintentionally. This spring plate 19makes it possible to block and prevent unintended opening of them.

FIG. 2 shows a more detailed view of the main card 4. The latterconsists of two portions of different thickness; on the left thethickness is greater and comprises the analysis zone 26. We also notethe presence of the drying stations 24, as well as a hole 31 forreceiving the distributor valve 22, which is not shown in this figure.Finally there are also six holes 32 for receiving the six pump pistons23, also not shown in this figure, within the main card 4. On theportion with small thickness of the main card 4, i.e. on the right ofthis figure, we note the presence of the storage zone 25 for thereaction liquid 12, after said reaction liquid 12 has:

-   -   resuspended the captured microorganisms 10,    -   provided lysis through ultrasonic agitation of the beads 6 via        this liquid 12,    -   transported the organisms of interest, and    -   finally supplied the reaction mixture 12 necessary for        amplification and detection of the nucleic acids 11.

FIG. 3 shows the secondary card 4. It is noted that on this portionthere are locations for receiving the O-ring seals 18 as well aslocations 33 for receiving the magnets 21; seals 18 and magnets 21 arenot shown in this figure.

FIG. 4 shows an enlarged view of a detail of FIG. 2, at the level of theretaining means 9 of the beads 6 as well as channels 7 and 8. The twoopposite ends of the C-shaped module are connected fluidically to thechannels for admitting and evacuating the reaction liquid 7 and 8.

In the present case, the means 9 consists of a grid 27, shown moreclearly in FIG. 5A or 5B. It is noted that the space separating theholes 28 of the grid 27 is small; for example for beads of 500 μm, theholes will have a diameter from 200 to 300 μm and will be separated fromone another by spaces of 200 to 300 μm, which leads to the fluids 2 orliquids 12 being forced into this space, which improves and increasesthe points of contact between the microorganisms, containing the nucleicacids of interest, and the surface of the beads 6 allowing bettercapture of said nucleic acids 11. The beads 6 are also coated with asubstance that further facilitates adhesion of the microorganisms on thesurface of the beads. This substance is notably glycerol in the casewhen the fluid used is a gas. Thus, the use of glycerol only functionsin the case of collecting microorganisms in a gas, notably air. In thisinstance, the air passes over the glycerol and the microorganisms arecaptured on the latter. The glycerol is then dissolved by the reactionbuffer 12. If bacteria are present, they will then be lysed in thisbuffer by ultrasound. When collecting microorganisms present in water,this does not function with glycerol, which is dissolved by water, andis driven out of the card. To achieve correct operation with a liquidmedium, a replacement must be found for glycerol, for example a polymercoating that does not dissolve in water but only in a specific reactionbuffer or allows “salting out” in this specific buffer, or a filter maybe added downstream of the beads so that the fluid comes into contactwith the beads before circulating through the filter.

According to FIGS. 5A and 5B, at the level of the main card 4, with thechamber or retaining means 9, it is noted that there are two ductsreferenced 40 and 41. Duct 40 allows admission of fluid 2 into themodule 3, said fluid 2 containing the bacteria to be captured forsubsequent analysis, whereas duct 41 allows evacuation of this samefluid 2 to the exterior of said module 3. Of course, between admissionand evacuation, the fluid 2 will be passed through the module 3 wherethe beads 6 are present; it is at this level that collection of themicroorganisms can be performed. If the fluid 2 is introduced andevacuated via ducts 40 and 41, the reaction liquid 12, which may consistof an elution buffer or other buffer, is introduced via an admissionchannel 7 into module 3, shown in FIG. 4, it is also evacuated from thissame module 3 via an evacuating channel 8, also shown in FIG. 44.

In fact this fluid 2, for example air, but it may also be a liquiddifferent from the reaction liquid 12, circulates perpendicularly to thecard. It flows through the grids 27.

FIGS. 5 to 8 give a simplified description of the protocol for captureof a microorganism.

FIG. 5A describes a portion of the device 1 according to the invention,at the level of treatment of the fluid 2, from which we wish to extractthe microorganisms 10, in which there are:

-   -   beads 6 present in the cavity, forming retaining means 9,    -   an upper grid and a lower grid 27, perforated by several holes        28, which confine the beads 6 within the device 1 and therefore        the retaining means 9 of the card 4,    -   a ferromagnetic top plate for confinement 15,    -   a ferromagnetic bottom plate for confinement 17, plates 15 and        17 not being glued to card 4 like the top and bottom plates for        confinement 14 and 16.    -   the fluidic channel, not shown in this figure. According to FIG.        5B, device 1 is more or less identical to the configuration in        FIG. 5A, but the ferromagnetic top plate for confinement 15 and        the ferromagnetic bottom plate for confinement 17 are translated        along F1. This allows the fluid 2 to pass, according to F2, into        the beads 6, held in position by the grids 27, by a movement        perpendicular to the channels for the reaction liquid or        liquids, not shown in the figure. This movement is performed via        the admission duct 40 and evacuating duct 41 of the fluid 2        within module 3.

FIG. 6 shows detail A from FIG. 5B. In this configuration, thedetachable confining plates 15 and 17 are removed according to F1, andthe fluid 2 circulates perpendicularly to the axis of the grids 27. Themicroorganisms 10 are captured on the beads 6 within the retaining means9.

In FIG. 7, the detachable plates 15 and 17 are returned to the closedposition by a sliding motion according to F3. The elution buffer 12 isintroduced into the chamber 9 via the admission channel 7. It detachesthe microorganisms 10 from the beads 6.

Finally in FIG. 8, once the chamber 9 is full, ultrasonic lysis iscarried out to disrupt the microorganisms, for example bacteria, 10 andrelease the nucleic acids 11. The lysate containing the nucleic acids 11is sent in the channels via the evacuating channel 8, for preparingamplification by resuspending the beads of lyophilized reagent, byoptical detection, etc.

The device therefore makes it possible to capture the microorganisms 10notably from the air on glycerol-coated glass beads by circulating theair stream through a bed of beads 6.

Capture of microorganisms by means of a dry surface offers manyadvantages, such as:

-   -   absence of wear of the medium during capture (due to        evaporation),    -   no evaporation during storage,    -   very high concentration of the microorganisms,    -   very large developed capture surface (due to the microbeads)        relative to a flat impacting surface,    -   the capture surface does not saturate so easily, capture is        therefore much more efficient than in an impaction system.

Moreover, the device is designed to:

-   -   offer a large surface area out of plane for capture of the        microorganisms from the fluid.    -   reduce the head losses and therefore the overall dimensions of        the pump; thus, during capture of airborne microorganisms, the        device is connected to a pump that takes in air through the        capture grid 27.    -   allow the microorganisms to be taken up in any liquid buffer        simply by pipetting owing to the use of fluidic channels in the        capture plane; thus, after capture of the microorganisms on the        glycerol, the detachable plates 15 and 17 are closed again. The        resuspension buffer (stored in chamber 25) is forced into the        chamber containing the beads. The microorganisms are then taken        in the glycerol; the latter dissolves in the buffer, so that the        microorganisms captured are released.    -   integrate lysis (notably with ultrasound) in the consumable or        device for collecting microorganisms 1,    -   obtain a high capture efficiency with a small head loss, as the        air passes over a bed of glycerol-coated beads (and is not        impacted on a bed of beads).

The capture efficiency largely depends on the thickness of the bed ofbeads and the size of the beads, but the capture efficiency issignificantly higher with capture in transmission through the beads,notably for particle sizes under 1 μm. This capture mode presentstechnical difficulties, such as:

-   -   control of the head loss,    -   keeping the beads in the capture chamber,    -   taking up the microorganisms in a buffer,    -   lysis.

Dry capture is performed without bubbling or aqueous gel, without lossof efficiency over time (>4 m³ sample). The advantage of glycerol isthat it does not dry out. Accordingly, the volume of air sampled can bevery large, without notable loss of efficiency. Systems with capture onaqueous surfaces (agar, Petri dish) or aqueous liquid (Coriolis type)have the drawback of drying out as capture proceeds. This drying causesa decrease in capture efficiency. Capture in one defined liquiddetermines the type of analysis performed downstream of collectionwhereas with dry capture the microorganisms may be taken up in anybuffer.

Moreover, capture on a dry surface (glass beads+glycerol) allows themicroorganisms to be released in a very small volume of buffer, lessthan 1 mL or even less than 100 μL. The advantage of increasing theconcentration of the microorganisms is that it promotes detection ofthem. The systems for detection in molecular biology have a limit ofdetection of the order of 1 genome equivalent per microliter (L).

With the conventional devices, it is therefore necessary to capture afar larger quantity (from 15 to 30 times more, i.e. from 200 to 600 μL)of microorganisms as the capture volume is 15 mL (large volume thatdilutes the microorganisms).

Mechanical lysis is very effective and is compatible with all types ofbiological methods, such as ultrasonic lysis in any buffer required forthe next steps of the protocol.

Lysis of the captured microorganisms is integrated in the device. Thelysis technique is purely mechanical (in contrast to chemical lysis)and, thus ensures an excellent lysis yield, regardless of the lysisbuffer selected and the microorganism to be lysed.

It is thus possible to use the lysate as it is, and then carry outamplification, for example NASBA or PCR.

According to a preferred use of device 1 proposed by the invention,after the microorganisms 10 have been lysed and the nucleic acids 11have been taken up in the reaction liquid or elution buffer 12, thecollecting device 1 makes it possible to perform amplification as wellas detection of said extracted nucleic acids 11. For this purpose, thereare mainly four steps that are carried out after lysis.

The first step consists of extracting the buffer 12 containing thenucleic acids 11 as well as a certain number of residues 30 ofmicroorganisms 10, which are present in the liquid 12 after lysis, theseresidues not being taken into account subsequently in the analyses thatwill be carried out at the level of the analysis zone 26. In this firststep, the liquid 12 laden with the nucleic acids 11 and the residues 30will exit from the retaining means 9 via the evacuating channel 8. Atthe level of the evacuating channel 8, it is for example possible tohave a filter that only allows biological elements to pass that have asize less than or equal to the nucleic acids extracted.

In a second step this mixture is sent into a zone for taking aliquots34, as clearly shown in FIG. 2. The volume thus obtained corresponds to120 μl.

This aliquot is then sent to a third step in a lysate separation zonereferenced 35. In this figure, there are six zones that are welldifferentiated from one another to allow separation in six times 20 μlin the analysis channels of zone 26. In these channels of zone 26,amplification is carried out, by means of reagents for amplification,such as nucleotides, amplification primers and detection probes, whichare positioned in the intermediate zone 36. It can be seen from FIG. 3that there is a second intermediate zone 37 containing at least oneenzyme (one enzyme if carrying out PCR, two if carrying out TMA andthree if carrying out NASBA), which is located on the front of the maincard 4.

The fourth and last step takes place at the level of the channels ofzone 26, as is clearly shown in FIG. 3 to the right of the intermediatezone 37 where detection may be performed.

All of these movements are therefore performed by means of thedistributor valve 22 and the pump pistons 23, which by their slidingmotion, not shown in the figures, make it possible to direct the liquidfrom the various zones to other zones that will allow amplification ofthe nucleic acids 11 and their subsequent detection. The distributorvalve 22 and the pump pistons 23 possess seals, clearly shown in FIGS.1, 38 and 39 respectively, which ensure hermeticity of the system.

Example 1: Efficiency of Capture of Microorganisms (with a ParticleCounter) as a Function of Bead Size and Thickness of the Bed of Beads

For one and the same device, here is the protocol for capture efficiencyas a function of bead size and thickness of the bed of beads.

1.A. Procedure:

The sliding plates 15 and 17 are open throughout the experiment. Forthis experiment, a particle counter is used which evaluates the quantityof particles per cubic meter (m³) of aspirated air. The particlesdetected are classified in different size categories between 0.3 μm, 0.5μm, 1 μm and 5 μm.

Three cycles of measurements are carried out:

-   -   1: Blank reference measurement without the device.    -   2: Evaluation measurement with the device upstream of the        particle counter.    -   3: A second reference measurement.

1.B. Experiment:

The blank reference measurements are averaged. The capture efficiency iscalculated as the ratio of the measurements with and without the device.

The results are shown in Table 1 below:

TABLE 1 Efficiency (in %) of capture of the microorganisms (with aparticle counter) as a function of bead size and thickness of the bed ofbeads Bead diameter Particle sizes Thickness of the bed of beads 0.3 μm0.5 μm 1 μm 5 μm 212-300 μm 50 87 95 98 1.5 mm 212-300 μm 65 85 92 952.5-3 mm 425-600 μm 18 65 87 95 1.5 mm 425-600 μm 45 85 90 89 2.5-3 mmWO-A-2009/001010 0 20 65 95

1.C. Analysis:

For one and the same device 1, Table 1 shows that the capture efficiencyis very effective regardless of the bead size and the thickness of thebed of beads relative to the prior art consisting of the devicedescribed in the PCT document. Particles smaller than 0.5 μm do notappear to give a sufficient yield, although it is still far higher thanthe prior art (minimum 18% yield versus zero). For a size between 0.5and 5 μm the results are good (always above 65% and even, if we excludethe case at 65%, always above 85%). All the thicknesses of the bed ofbeads tested are acceptable.

Example 2: Optimization of the Head Loss of Each of the Devices Tested

2.A. Procedure:

Each device is placed on a test bench, allowing the head loss to bemeasured as a function of the air flow rate. The objective is to findthe best compromise between capture efficiency of the device 1 and itshead loss. A high head loss signifies high electrical consumption of thesampling device (not favorable for portable applications).

2.B. Experiment:

Table 2 is a table for aiding design of the device for capture ofmicroorganisms. It compares the particle capture efficiency (by sizerange: 0.5-1 μm, 1-5 μm, 5-25 μm) with the head loss (mbar) of thedevice. The energy required for pumping a certain volume of air throughthe device is proportional to the head loss (for one and the same flowrate). For application with a portable pump it is necessary to find agood compromise between the capture efficiency and the head loss (seeTable 2).

TABLE 2 Efficiency of capture of microorganisms by particle size rangesand head loss of the device tested (at 50 L/min) as a function of beadsize and thickness of the bed of beads Particle sizes Bead diameter0.5 - 1 - 5 - Head loss Thickness of the bed of beads 1 μm 5 μm 25 μm(mbar) 212-300 μm 75 95.5 95 15 1.5 mm 212 -300 μm 80 92 95 25-30 2.5-3mm 425-600 μm 56 89 93 6-7 1.5 mm 425-600 μm 72 89 88 15 2.5-3 mmWO-A-2009/001010 43 80 94.5 6-11

2.C. Analysis:

In light of this table and in comparison with patent applicationWO-A-2009/001010, constituting the prior art, it can be seen that thedevice according to the invention, regardless of the thickness of thebed of beads and the diameter of the beads used, always gives betterperformance than the solution proposed by the prior art. Moreover, withbeads from 425 to 600 μm and a bed thickness of 1.5 mm, the results arethe most efficient for capturing the particles than the device of patentWO-A-2009/001010 for an equivalent head loss.

Example 3: Filling and Emptying the Devices

3.A. Procedure:

Only the chamber 9 of beads 6 is tested. The inlet of the bead chamber(full of beads but without liquid) is connected to a fluidic connectorand a pipette. The liquid is forced into the bead chamber until it isfull. Once filled, it is emptied in the same way.

3.B. Experiment

FIGS. 10A to 10D show filling of the chamber 9 where the microorganisms10 are captured. 500 μL is injected at the level of the admissionchannel 7 and 200 μL is recovered at the level of the evacuating channel8.

3.C. Results:

Filling takes place perfectly.

Example 4: Lysis of the Microorganisms

4.A. Procedure:

A device 1 consisting of a lysis chamber is filled with a buffer 12containing Staphylococcus epidermidis. The device is subjected toultrasound, via a sonotrode, in order to lyse the microorganisms. Theexperiment is repeated with a lysis time of 0, 1, 5 and 10 minutes. Thelysis yield is evaluated by growth and counting of the lysates on aPetri dish. The objective of this experiment is to find a suitableinterface for transmission of ultrasound in the lysis step.

4.B. Experiment:

In relation to FIG. 11, one and the same design of bead chamber deviceis used with some interface configurations, i.e.:

-   -   1st configuration: with plastic elements replacing the        ferromagnetic plates 15 and 17 (sonotrode in direct contact with        a modified device according to the invention),    -   2nd configuration: with a layer of silicone (present between the        sonotrode and the device according to the invention),    -   3rd configuration: with metallic elements serving as        ferromagnetic plates 15 and 17 added to the device.

The three devices are tested as before and for two types of beaddiameter (see Table 3).

TABLE 3 Efficiency of ultrasonic lysis Silicone Device Standard Metalinsert layer Diameter of 212-300 425-600 212-300 425-600 212-300 glassbead (μm) Lysis  90-100 50 100 98 40-55 efficiency (%)

4.C. Results:

The results are quite good for all of the tests carried out. However,the solution using the metal inserts gives good results that are more orless identical for both types of beads.

Example 5: Lysis and NASBA Amplification without Purification ofMicroorganisms (S. epidermidis)

5.A. Procedure:

A device is filled with buffer containing S. epidermidis bacteria aswell as an internal control consisting of a bacterium different from thebacterium tested. Lysis is carried out in the device according to the3rd configuration described in example 4.

The lysate is then removed from the device and analyzed.

5.B. Experiment:

The detection curves are presented in Tables 4 and 5 below.

Reference (control range) (eq. CFU/μl) 0 0.1 1 10 100 1000 10000 ResultNeg- Pos- Pos- Pos- Pos- Pos- Pos- ative itive itive itive itive itiveitive

Samples tested 0 0.2 2 20 200 (eq. CFU/μl) Result Negative PositivePositive Positive Positive

This experiment shows the efficiency of the device described for lysingthe microorganisms and for detecting them by NASBA analysis. Themicroorganisms are injected into the buffer 12 before the experiment (adifferent concentration of microorganism in each experiment). The metalflaps of the card are closed. The part containing the beads is filledwith buffer 12 (which contains various concentrations ofmicroorganisms). The device is placed on an ultrasound probe to performlysis. At the end of the lysis step, the buffer 12 is collected manuallyand analyzed by NASBA away from the card. These results are to becompared with a control range.

By first intention, the interpretation of the results is binary:Positive signifies that there is detection of the microorganism andnegative signifies that the microorganism is not detected.

5.C. Analysis:

It can be seen from the above tables that the samples tested are markedas positive for all concentrations above 0.2 CFU/μl.

REFERENCE SYMBOLS

-   -   1. Device for collecting microorganisms 10    -   2. Fluid    -   3. Reaction module    -   4. Main card    -   5. Secondary card    -   6. Beads    -   7. Reaction liquid admission channel 12 of module 3    -   8. Channel for evacuating the reaction liquid 12 from module 3    -   9. Retaining means of the beads 6 within module 3    -   10. Microorganisms present in the fluid 2    -   11. Nucleic acids present in the microorganisms 10    -   12. Reaction liquid    -   13. Airtight confinement enclosure consisting of plates 14 to 17    -   14. Top plate for confinement of device 1    -   15. Sliding ferromagnetic top plate for confinement of device 1    -   16. Bottom plate for confinement of device 1    -   17. Sliding ferromagnetic bottom plate for confinement of device        1    -   18. Hermetic O-ring seals between the enclosure 13 and the cards        4 and 5    -   19. Spring plate for locking the ferromagnetic plates 15 and 17    -   20. Fixing screw of the spring plate 19    -   21. Magnets    -   22. Distributor valve    -   23. Pump pistons    -   24. Desiccating stations    -   25. Storage zone of reaction liquid 12    -   26. Analysis zone on the front and back of the card    -   27. Upper and lower grids    -   28. Holes in grid 27    -   29. Slides    -   30. Residues of microorganisms present in the liquid 12 after        lysis    -   31. Hole for receiving the distributor valve 22    -   32. Holes for receiving the pump pistons 23    -   33. Through-holes of cards 4 and 5 receiving the magnets 21    -   34. Aliquoting zone    -   35. Separation zone    -   36. First intermediate zone containing an enzyme    -   37. Second intermediate zone containing an enzyme    -   38. Seals on the distributor valve 22    -   39. Seals on the pump pistons 23    -   40. Fluid admission duct 2 within module 3    -   41. Duct for removing the fluid 2 within module 3    -   F1. Sliding motion of the ferromagnetic top plate 15 and        ferromagnetic bottom plate 17 allowing opening for passage of        fluid 2    -   F2. Movement of fluid 2 passing through beads 6    -   F3. Sliding motion of the ferromagnetic top plate 15 and        ferromagnetic bottom plate 17 allowing closure to passage of        liquid 12    -   F4. Movement of liquid 12 passing through beads 6    -   F5. Extraction of the nucleic acids 11

1. A method for collecting microorganisms when contained in a fluid,comprising: introducing the fluid into a cavity of a collecting devicevia at least one admission duct; capturing the microorganisms whencontained in the fluid with a set of beads retained in the cavity as thefluid passes through the set of beads; evacuating the fluid from thecavity via at least one evacuating duct; introducing a reaction liquidinto the cavity via at least one admission channel; collecting themicroorganisms from the set of beads with the reaction liquid as thereaction liquid passes through the set of beads; and evacuating thereaction liquid from the cavity via at least one evacuating channel. 2.The method as claimed in claim 1, wherein the admission channel andevacuating channel and the admission duct and evacuating duct arepositioned as follows: the admission and evacuating ducts face oneanother along an axis; the admission and evacuating channels arepositioned respectively at two opposite ends of the cavity; and theadmission and evacuating channels are positioned in a planeperpendicular to the axis.
 3. The method as claimed in claim 1, whereinat least one upper plate and at least one bottom plate of the collectingdevice are configured to close a reaction module including the cavity toform an airtight confinement enclosure in order to isolate the fluid andthe reaction liquid from outside the collecting device.
 4. The method asclaimed in claim 2, wherein the cavity has a “C” shape if the cavity iscut along the plane in which the admission and evacuating channels arepositioned, to ensure that the reaction liquid passes completely throughthe set of beads between a moment when it is admitted and a moment whenit is evacuated.
 5. The method as claimed in claim 4, wherein the cavityhas a quadrilateral shape if the cavity is cut along a radius passingthrough the center of the “C” shape and cutting through the “C” shape.6. The method as claimed in claim 4, wherein the admission channel isconnected to one end of the “C” shape of the cavity, and the evacuatingchannel is connected to the other end of the “C” shape.
 7. The method asclaimed in claim 1, wherein the beads have a diameter in the range from200 to 600 μm and are retained in the cavity by retaining elements thatcomprise grids including pores having a diameter smaller than thediameter of the beads and in the range from 100 to 500 μm.
 8. The methodas claimed in claim 1, wherein the fluid is a gas, the set of beadsincludes beads coated with glycerol, and the reaction liquid liquefiesthe glycerol upon contacting the beads coated with glycerol.
 9. Themethod as claimed in claim 1, wherein the fluid is a liquid and passesthrough the set of beads and then a filter before being evacuated. 10.The method as claimed in claim 1, wherein the reaction liquid isfiltered between being evacuated via the evacuating channel and beingprovided to a reaction module configured to separate, amplify, and/ordetect nucleic acids.
 11. The method as claimed in claim 1, wherein thebeads are agitated to lyse membranes of the microorganisms as thereaction liquid passes through the set of beads.
 12. The method asclaimed in claim 11, wherein the beads are agitated by ultrasound. 13.The method as claimed in claim 1, further comprising separating nucleicacids from cellular residues of the microorganisms in the collectingdevice.
 14. The method as claimed in claim 13, further comprisingamplifying the nucleic acids to obtain amplicons and detecting theamplicons in the collecting device.